Manufacture of polysaccharide beads

ABSTRACT

The present invention is a process of manufacture of one or more polysaccharide beads, comprising generating an aerosol of an aqueous polysaccharide solution, cooling the droplets of said aerosol in air to initiate gelling thereof and collecting droplets as gelled beads in a liquid or on a surface, characterised by adding a hydrophilic vapour pressure-lowering agent to said polysaccharide solution.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a filing under 35 U.S.C. 371 and claims priority tointernational patent application Ser. No. PCT/SE03/00629 filed Apr. 17,2003, published on Nov. 6, 2003 as WO 03/091315 and also claims priorityto patent application Ser. No. 0201289-6 filed in Sweden on Apr. 25,2002; the disclosures of which are incorporated herein by reference intheir entireties.

TECHNICAL FIELD

The present invention relates to the manufacture of microspheres, andmore specifically to the manufacture of porous polysaccharide beads. Theinvention also relates to porous polysaccharide beads as such.

BACKGROUND

Porous microspheres have found use for many purposes, such as supportfor growth of micro-organisms and as carriers in separation techniques.Gelled microspheres have been shown to exhibit especially advantageousproperties in chromatographic separations, for example as concerns masstransport, and are therefore one of the most widely used carriermaterials at present.

Conventionally, polysaccharide beads have been produced by inversesuspension techniques. In brief, such methods use a heated aqueoussolution, which includes a thermally gelling polysaccharide. Saidsolution is mixed into a heated organic solvent, such as toluene. Theaqueous solution and the solvent form an emulsion, which is then cooledto allow the aqueous phase of the emulsion to gel into the form ofmicrospheres. However, the hazardous nature of the solvents normallyused renders them undesired to handle, since in general they are oftenneurotoxic and also highly flammable. In addition, extensive washingprocedures are required to remove the solvent before use of theparticles e.g. in chromatography. Furthermore, inverse suspensiontechniques usually result in particle populations of relatively broadsize distributions. Since many practical applications require particlesof a similar or almost identical size, an additional step e.g. bysieving is then required after the inverse suspension step. Accordingly,these techniques are also time-consuming and consequently costlyprocedures.

To avoid the above-described disadvantages, polysaccharide beads havemore recently been manufactured in methods wherein beads are formed andgelled in air rather than in a solvent. For example, U.S. Pat. No.6,248,268 (FMC Corporation) describes how polymer microparticlessuitable for gel chromatography are formed by spraying a composition ofa thermally-gelling polymer in an aqueous medium into ambient air andallowing the atomised composition to gel in the air. If a rehydrateablemicrogel is desired, then a non-gelling hydrocolloid is added. Suchhydrocolloids are exemplified by a number of various polymers, forexample various polysaccharides and some synthetic polymers.

However, a drawback with beads so produced is that the water evaporationfrom the droplets will result in a skin on the bead surface with lowerpore size than the bulk of the bead. The skin will restrict the masstransport properties of the bead and often needs to be compensated byuse of a lower agarose concentration in the bead-forming solution.However, such a low agarose concentration will in turn result in poormechanical properties of the beads.

The formation of skin in the field of polymer microparticles has beendiscussed in U.S. Pat. No. 6,248,268 (XC Corporation). However, thediscussed skin is not a skin on the particle surface, instead U.S. Pat.No. 6,248,268 suggests how it can be avoided that the particles flattenout and form a skin on the surface of an aqueous medium used forcollecting the microparticles. More specifically, it is disclosed how itis advantageous to avoid dehydration of the formed particles by allowingthem to cool in water rather than in air. It is in this context it issuggested to add a surfactant to avoid that all sprayed particles arecollected on the surface of said cooling water.

WO 02/12374 (Prometic Biosciences) discloses an apparatus that includesa rotating atomiser wheel onto which a uniform thin layer of a polymeris applied via a distributor. Due to the centrifugal force, the polymerwill move to the periphery of the wheel and free flying particles aresubsequently released. The apparatus further includes a catch tray tocollect the porous polymer particles produced and an enclosure defininga partition between an interior environment and an exterior environmentof the apparatus. Within the interior, the temperature and humidity areclosely controlled so as to produce beads of narrow particle sizedistribution. However, even with this technology, the beads producedhave been shown to exhibit the above-discussed undesired skin.Accordingly, controlling the humidity surrounding the beads is not asufficient solution for solving the problem of a too rapid evaporationfrom polysaccharide beads as produced with spinning disk technology.

Thus, there is still a need in this field of new methods for producingpolysaccharide beads with advantageous mass transport and mechanicalproperties to reasonable costs.

SUMMARY OF THE PRESENT INVENTION

One object of the present invention is to provide a process ofmanufacture of polysaccharide beads, which process results directly in apopulation of beads exhibiting a sufficiently narrow size distributionto avoid additional steps of sieving. Accordingly, the present processis a continuous and cost-efficient process. The polysaccharide beadsmanufactured according to the invention are useful e.g. in separationmethods, either directly as in gel diffusion chromatography, hydrophobicinteraction chromatography (HIC), or reverse phase chromatography (RPC),or, after suitable derivatisation, in affinity or ion exchangechromatography (IEX).

Another object of the present invention is to provide a process ofmanufacture of polysaccharide beads, which process avoids the problem offormation of skin on bead surface caused by undesired water evaporation.

A further object of the invention is to provide a process as describedabove, which results in beads that are advantageously used aschromatographic carriers due to improved mass transport properties and agood mechanical strength.

An additional object of the invention is to provide polysaccharide beadshaving one or more of the above discussed properties of a narrow sizedistribution, an eliminated or at least reduced surrounding skin andadvantageous mechanical strength and mass transport properties. Aspecific object of the invention is to provide one or more such beads,which has also been derivatised with ion exchanging groups, which beadscan be used in a conventional chromatographic separation withoutcracking due to the osmotic pressure induced by the fixed charges.

One or more of the above-described objects are achieved according to theinvention as defined in the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an example of equipment suitable for manufacture of beadsaccording to the present invention.

DEFINITIONS

In the present specification, the term “bead” is used for essentiallyspherical microspheres, and refers to particles the diameter of which isin the region of up to about 500 μm.

The term “aerosol” refers to a multitude of droplets, individuallysurrounded by air or any other gas.

A “surfactant” is defined herein as a chemical compound, whichdrastically lower the surface tension of water even at a lowconcentration and which comprises one hydrophobic and one hydrophilicpart.

DETAILED DESCRIPTION OF THE INVENTION

Thus, a first aspect of the invention is a process of manufacture of oneor more polysaccharide beads, comprising generating an aerosol of anaqueous polysaccharide solution, cooling the droplets of said aerosol inair to initiate/allow gelling thereof and collecting droplets as gelledbeads in a liquid or on a surface, characterised by adding a hydrophilicvapour pressure-lowering agent to said polysaccharide solution.

The solution of polysaccharide is an aqueous solution, which isconveniently obtained by adding polysaccharide to water, an aqueous saltsolution or the like. However, excessive amounts of salt should beavoided, since it may impair the final pore structure of the bead.Mixing is preferably performed at an increased temperature, such as afew degrees below the boiling point of the solution, which willfacilitate the dissolution. In general, the temperature of the water oraqueous solution should be kept low, such as below about 40° C., toavoid formation of lumps. Heating of the polysaccharide solution can beprovided by any conventional means, such as by water bath, microwaveoven etc.

As regards the vapour pressure-lowering agent, the requirement thereofis a capability to lower the vapour pressure of the polysaccharidesolution. As the skilled person in this field will know, vapour pressurelowering is a colligative property and can depend on factors such as themole fraction of the additive, size etc. For example, polymers are ingeneral known to be poor vapour-pressure-lowering agents and are forthat reason avoided according to the present invention. Accordingly, thehydrocolloids added according to the above-discussed U.S. Pat. No.5,662,840 are all polymers and are, as the skilled person in this fieldeasily realises, consequently too large to be capable of efficientlylowering the vapour pressure in an aqueous polysaccharide solution.Further, the present vapour pressure-lowering agent will also exhibit aboiling point, which is sufficiently high to essentially avoid or atleast to substantially reduce evaporation from the surface of thegelling droplets. As mentioned above, the polysaccharide solution isaqueous and the nature of the vapour pressure-lowering agent shouldconsequently be sufficiently hydrophilic to dissolve therein. As theskilled person in this field will realise, the vapour pressure-loweringagent should also be selected so that it does not have any negativeimpact on the gelling properties of the agarose.

Accordingly, in one embodiment of the present process, the vapourpressure-lowering agent comprises molecules of a Mw below about 200 D,preferably below about 190 D, more preferably below about 150 D and mostpreferably below about 80D, and is defined by a vapour pressure belowthat of water, such as below about 310 mbar, preferably below about 250and most preferably below about 200 mbar at 70° C. Using anotherdefinition, in one embodiment the present process uses a vapourpressure-lowering agent that will result in a surface tension no lowerthan 50 mN/m at 10 wt % concentration in water at 25° C.

The reason for keeping the surface tension above said value is to avoidthe formation of small satellite droplets that can give an undesirablywide bead size distribution. Hence, it is also important that anyfurther additives do not lower the surface tension much further. It caneven be advantageous if the surface tension is somewhat increased.However, in the present context, it is noted that measurements ofsurface tension in a complex solution such as the polysaccharidesolution used herein can be complicated due to its viscous nature andthe strong tendency for evaporative skin formation against air surfacesat elevated temperatures.

Alternatively, the nature of the vapour pressure-lowering agent isdefined by a boiling point which is at least about 130, such as about135, preferably at least about 150° C., such as about 153° C., and mostpreferably at least about 190° C., such as about 197° C.

Thus, the present invention shows for the first time that by adding asuitable amount of a vapour pressure-lowering agent to thepolysaccharide solution, beads of an unexpected quality as concerns thesize of surface pores and consequently mass transport can be obtained.Even though efforts have been made in the past to manufacture such beadsfrom polysaccharide aerosols gelled in an atmosphere of increasedhumidity the beads so obtained have exhibited the above-discussedundesired skin that partly or fully occludes pores on the bead surface.Accordingly, it was quite unexpected to find that addition of a vapourpressure-lowering solvent could indeed reduce and even eliminate suchskin formation.

In one embodiment, the vapour pressure-lowering agent is an organicsubstance. Thus, in a specific embodiment, the vapour pressure-loweringagent is selected from the group that consists of glycols, such asethylene glycol, propylene glycol (i.e. propane-1,2-diol),propane-1,3-diol, butane-1,2-diol, butane-1,3-diol, butane-1,4-diol,butane-2,3-diol, diethylene glycol, dipropylene glycol and triethyleneglycol; glycerols; such as glycerol, glyceryl ethers or glyceryl esters;polyols such as sorbitol, mannitol, glucose, sucrose, trimethylolpropaneor pentaerythritol; amides, such as carbamide (urea), formamide,dimethylformamide, acetamide, dimethylacetamide or pyrrolidone; ethers;carboxylic acids; esters; alcohols; organosulfides; organosulfoxides,such as dimethylsulfoxide; sulfones, such as dimethyl sulfone; alcoholderivatives; glycol ethers; such as butyl carbitol or cellosolve; etherderivatives; amino alcohols; and ketones. In a specific embodiment, thevapour pressure-lowering agent is selected from the group that consistsof glycols, such as ethylene glycol or propylene glycol; and glycerol.In an advantageous embodiment, the vapour pressure-lowering agent isethylene glycol.

As a general rule, the amount of added vapour pressure-lowering agentshould not exceed about 50% and is preferably below about 35%, such asbelow about 30%. Expressed as intervals, the amount of added vapourpressure-lowering agent is 0-50%, such as 0-35% and specifically 0-30%.

In an advantageous embodiment of the present process, the aerosol isgenerated by a spinning or rotating disk technique. The basic principlesof such rotary atomiser machines have been described in general, seee.g. in Spray Dying Handbook, K. Masters, 5th ^(e)d., Longman Scientific& Technical, Longman Group UK Limited; and Atomization and Sprays, A.Lefebvre, Hemisphere Publications, 1989; and Liquid Atomzation, L.Bayvel and Z. Orzechowski, Taylor and Francis, 1993. A specific exampleis also described in WO 88/07414 (Prometic Biosciences Inc). However, inalternative embodiments, the aerosol is generated by any otherconventional means, such as spraying, see e.g. U.S. Pat. No. 6,248,268.The equipment for manufacture of the beads is advantageously controlledby suitable software, as is well known in this field.

In one embodiment, droplets are subsequently collected on a surface,such as a sloped surface, which is preferably coated with a film ofwater. In the preferred embodiment, the droplets are collected in a bathcomprised of a liquid having a reduced surface tension, such as waterwith an added surfactant. The recovered beads can be stored assuspensions or slurries.

In an especially advantageous embodiment of the present process, thepolysaccharide droplets are cooled in an essentially steam-saturatedatmosphere. To enable control of the surrounding atmosphere, theabove-discussed apparatus can be provided with a housing or enclosurearranged over the spinning disk.

The content of polysaccharide in the solution from which the aerosol isgenerated should be kept at a level that enables for the droplets toretain their structural integrity as they travel through the air andreaches the surface or liquid. The polysaccharide present in the aerosoldroplets will begin to gel as soon as a temperature below its gellingtemperature is reached. During the gelling, a physical cross-linking ofthe polysaccharide will occur, resulting in a porous bead. The solidcontent of the beads produced according to the invention will be in therange of about 0.5-15%, preferably about 2-12 and most preferably about4-6%. As the skilled in this field will understand, the viscosity of thesolution will decide the upper useful limit, and accordingly, a higherpolysaccharide content can at least in part be compensated by loweringthe molecular weight of the polysaccharide and/or by intensifying theatomisation conditions. Similarly, as the skilled person will realise,the lower figures of the interval given above may be compensated by asuitable additive.

For some applications, it is desired to further increase the mechanicalstability of the manufactured bead. For example, a more rigid bead willbetter withstand high column pressures when used in chromatography.Accordingly, in one embodiment of the present process, thepolysaccharide solution also comprises an added cross-linker. Thecross-linker will provide a chemical cross-linking in addition to thephysical cross-linking that occurs spontaneously during gelling. Thereis a large number of conventionally used and well known cross-linkersavailable, and accordingly the skilled person in this field can easilyselect a suitable one depending on the polysaccharide used, the intendeduse of the final product etc. As illustrating examples, epichlorohydrin,divinylsulfone, di- or polyglycidyl ethers, alkyl dihalogenides,polysaccharides polysubstituted with reactive groups (e g allyl agarose)etc.

In specific embodiments, one or more further components are added to thepolysaccharide solution before gelling. Such components are e.g.components valuable for performing ion exchange or affinitychromatography using the final bead, one or more polymers, granules,high density particles, magnetic particles, fibres, leachable templateparticles etc.

In one embodiment of the present process, the polysaccharide is selectedfrom the group that consists of agarose, alginate, carrageenan,furcelleran, gellan, konjac, pectin, curdlan, starch and galactomannans.Such polysaccharides are known to form physically cross-linked networksspontaneously on cooling or on addition of divalent metal ions. In apreferred embodiment, the polysaccharide is agarose, such as ahigh-viscosity or low-viscosity agarose. In the case of agarose beingthe polysaccharide, which can be cross-linked according to conventionalmethods after the droplet formation with epichlorohydrine. In the caseof allyl agarose, cross-linking can be performed in a sequence of stepsincluding addition of epichlorohydrine and salt and bromation of theallyl groups, also in accordance with well-known methods.

A second aspect of the present invention is a population ofpolysaccharide beads produced from an aerosol generated from apolysaccharide solution and cooled in air, wherein regional dehydrationin surface layer has been totally or at least almost totally avoided. Asshown in the experimental part below, this is evidenced by a Kav-valuewhich is increased as compared to the Kav-value for a corresponding beadprepared with equivalent process parameters but without use of anyvapour pressure-lowering agent. (Porosity values and/or pore sizes ofpolysaccharide beads are often expressed as exclusion limits in terms ofhow large portion of the material a particular compound can utilise(Kav). See Hagel in “Protein Purification, Principles, High Resolution,and Applications”, J-C Janson and L Rydén (Eds), VCH Publishers Inc. NewYork, 1989, p. 99.)

The polysaccharide beads according to the invention can also becharacterised as beads with mass transport properties and mechanicalstrength comparable to polysaccharide beads produced according toconventional emulsion technologies, while they contain no traces of thesolvent used in said emulsification, such as toluene.

In one embodiment, the population of polysaccharide beads according tothe invention has been produced according to a process of manufacture asdefined above.

In another embodiment, the polysaccharide bead or population ofpolysaccharide beads according to the invention have further beenderivatized with affinity or ion exchanging groups. Derivatisation ofagarose beads is easily done by the skilled person in this field inaccordance with standard procedures.

DETAILED DESCRIPTION OF THE DRAWINGS

FIG. 1 shows schematically an apparatus suitable for use of the methodaccording to the present invention. More specifically, FIG. 1 shows agel pump 1 for feeding gel, via gel feed valve 12 and gel gun and valve15, onto spinning disc(s) 5. The apparatus is provided with a vapourgenerator 2 for producing vapour that can be fed via upper vapour valve6, and lower vapour valve 7 into a dome 9. The apparatus has an upperpart 3, which is adjustable up and down, and a lower part 4 with a motor8 for spinning disc(s) 5, a catch 10 for collecting particles spun offdisc(s) 5 and a regulator 11 for controlling the fluid level in catch 10which delivers the particles to a sieve 14 for particle concentration.The apparatus is also provided with a cleaning liquid tank 13 forcleaning of the apparatus.

Experimental Part

Below, the present invention will be illustrated by way of examples,which are not to be construed as limiting the scope of the presentinvention as defined in the appended claims. All references given belowand elsewhere in the present specification are hereby included herein byreference.

EXAMPLE 1 Agarose Beads (5%) Prepared According to the Invention

Agarose solution: 82.5 g of agarose, 525 g of propylene glycol and 2.75g of KHPO₄ were dissolved in 1000 g of water at 100° C. The temperatureof the solution was allowed to decrease a few degrees and then boilingwas performed again. The solution was transferred to an autoclave andleft at 1 standard atmosphere for 2×15 minutes, after which water wasadded to compensate for steaming during the preparation. The temperaturewas then lowered to 58° C., which was the temperature of manufacture.

Manufacture of beads: The gel prepared as described above was heated to95° C. for 120 minutes, the temperature was lowered to 70° C. Theequipment described in FIG. 1 was controlled from possible remains oflast run, plastic folio was put over the centre to protect fromwater/humidity, and the plastic folio skirt was controlled for cleannessand length, i.a. the spaltum width. The system was then washed throughwith water and air, and it was checked that all valves were working. Thegel and steam delivery systems were then assembled. The discs wereassembled and centricity adjusted. The system was tested by a lowvelocity dry run and checked for wobbling. The gel pump (MELTEX, typePUR103-1, no C18160987, 220 VAC, 4200W) was equilibrated, the hose andthe gel delivery system were provided with thermostat.

Water was pumped through the system, with plastic cover over the lowercentre max flow. The catch water system drain was started direct tocesspool, the sieves cleaned and then a suitable set of sieves wereassembled. The plastic cover over the lower centre was removed.

The disc rotation was started at 55 Hz (RotorDisque), the detergent pumpwas started, and the control computer program was started. The steamcondensate traps were flushed, the individual steam needle valvesettings were checked, the gel pump was started, with water, and thesystem temperature was allowed to equilibrate for 10 min, while theoutlet of the catch was allowed to pass the sieves.

The gel pump was then almost emptied of water, but care was taken not tolet it go dry. Then the dissolved agarose gel, prepared as describedabove, was added and the disc rotation was increased to 83 Hz. Steamcondensate traps were flushed every 30 minutes.

EXAMPLE 2, COMPARATIVE Agarose Beads (5%) Prepared Without anyVapour-pressure Lowering Agent

For the preparation of agarose solution, the procedure of example 1 wasfollowed, except that here the addition of propylene glycol was omitted.The beads were manufactured as described in example 1.

EXAMPLE 3 Agarose Beads (6%) Prepared According to the Invention

Preparation of agarose solution: 99 g of agarose and propylethylene to aconcentration of 30% were dissolved in 1500 g of water at 100° C. Thetemperature of the solution was allowed to decrease a few degrees andthen boiling was performed again. The solution was transferred to anautoclave and left at 1 standard atmosphere for 2×15 minutes. 3 ml ofglacial acetic acid was added at 75° C., and the solution was left withstirring for 45 minutes to hydrolyse, which was interrupted by additionof 26 g of KHPO₄ (0.2 mole) dissolved in 50 ml water at 70° C., whichwas the temperature of manufacture.

The beads were manufactured as described in example 1.

EXAMPLE 4, COMPARATIVE Agarose Beads (6%) Prepared Without any VapourPressure-lowering Agent

For preparation of the agarose solution, the procedure of example 3 wasfollowed, except that here the addition of propylene glycol was omitted.The beads were manufactured as described in example 1.

RESULTS Ex. 1 Ex. 2 Ex. 3 Ex. 4 Prod.parameters: Experiment: GlycolComp. Glycol Comp. Raw material lit.: 1.5 1.5 1.5 3 Gel concentration %:5 5 6 6 Gel pump l/h: 1.5 1.5 1.5 1.5 Temp. bowl ° C.: 58 57 73 67 Temphose ° C.: 60 58 72 65 Temp. nozzle ° C.: 60 58 72 65 Temp. gel fromnozzle ° C.: 60 58 70 65 Temp. gun ° C.: 62 60 68 79 Temp. valve ° C.:63 61 71 70 o m i o m i o m i o m i Steam over: 0.3 0.3 0.3 0.3 0.3 0.30.3 0.2 0.2 0.2 0.2 0.2 Steam under: 0.2 — 0.2 0.2 — 0.2 1.0 — 0.2 0.2 —0.2 Steam pressure bar: 0.75 0.75 0.75 0.75 Droplet generator r/m: 53005000 5000 6000 Process data, result: Top, steam collar ° C.: 110 107 9490 Middle over disc ° C.: 75 67 56 68 Bend before nozzle ° C.: 60 58 6863 Dome ° C.: 38 40 36 42 Crate ° C.: 21 22 20 24 TS %: 4 4.6 4.5 4.9Porosity Ve/V0: 1.57 Kav, Thy: 0.17 0 0.33 0

1. In a process of manufacture of one or more polysaccharide beads,comprising generating an aerosol of droplets of an aqueouspolysaccharide solution in air, cooling the droplets of said aerosol inair to initiate gelling thereof and collecting droplets as gelled beadsin a liquid or on a surface, the improvement comprising adding ahydrophilic vapour pressure-lowering agent to said polysaccharidesolution to reduce skin formation on the bead surface.
 2. The process ofclaim 1, wherein the vapour pressure-lowering agent includes moleculesof a molecular weight below about 200 D, and is defined by a vapourpressure below about 310 mbar at 70° C.
 3. The process of claim 1,wherein the vapour pressure-lowering agent results in a surface tensionabove about 50 mN/m at 10 wt % concentration in water at 25° C.
 4. Theprocess of claim 1, wherein the vapour pressure-lowering lowering agentis an organic substance.
 5. The process of claim 1, wherein the vapourpressure-lowering agent is selected from the group consisting of glycolsand glycerols.
 6. The process of claim 5, wherein the vapourpressure-lowering agent is propylene glycol.
 7. The process of claim 1,wherein the aerosol is generated by a spinning disk technique.
 8. Theprocess of claim 1, wherein the aqueous polysaccharide droplets arecooled in an essentially steam-saturated atmosphere.
 9. The process ofclaim 1, wherein the polysaccharide solution includes a chemicalcross-linker.
 10. The process of claim 1, wherein the polysaccharide isagarose.